The present invention relates to a method as well as a device for identification of a substance, preferably comprising biochemical molecules. In particular, it is the aim of the present invention to improve the technique of analyzing unknown biomolecules for achieving results in an easier and quicker way so that the method can be used without investing great effort and high costs. A preferred embodiment of the invention is a biosensor for easy detection of unknown biomolecules.
Many approaches have been undertaken for determining the type of substance such as a chemical substance, in particular a biomolecule, which provides a very high degree of selective binding properties.
There are methods of analyzing the optical properties of individual substances such as absorption or transmission behavior, all these methods require very accurate measurement equipment comprising light sources for emitting defined wavelengths as well as very accurate detection devices, rendering these methods very expensive:
Methods are known for identification of substances by measuring their surface properties such as surface forces or work of adhesion. The work of adhesion of a substance coated onto a solid surface can be determined by measuring the contact angle between the surface and a fluid droplet applied onto it. This technique is described in an article by Israelachvili, ,xe2x80x9cInterfacial Forcesxe2x80x9d, J.Vac.Sci. Technol. A 10(5), September/October 1992, pp. 2961-2971.
Another approach for estimating the surface free energies of solid surfaces requires direct examination of solid-solid interfaces. In an article by Chaudhury and Whitesides, Langmuir, vol. 7,. No. 5, 1991, pp. 1013-1025, measurement of the surface free energy of elastomeric polymers is deduced by measurement of the deformation of semispherical lenses of poly-dimethylsiloxane elastomers and their chemical derivatives. Analyses of the deformation of the lens were conducted using Young""s equation and the Johnson, Kendall Roberts (YRK) model. The analyses included balancing the adhesion forces acting across the interface with the restoring forces that oppose the deformation of the lens. Accurate results require the Young""s modulii and the Poisson ratios of each material. In addition, spherical lenses with a well-defined radius and smooth surface have to be produced for one or both materials involved in the measurement.
By determining the work of adhesion of unknown substances which are derivatized onto a sample surface, an estimation can be made about the nature and the type of the derivatized substance. However, the aforementioned methods are complicated and drawn out.
Another aspect in determining binding constants of substances by measuring their surface properties is the direct interaction between at least two molecules. Such interactions, derived from multiple weak bonds between geometrically complementary surfaces which are coated with different chemical groups, can be very strong. In the field of biomolecules, the sensitivity of binding properties is very distinct so that molecular recognition between a receptor and a ligand, an antibody and an antigen as well as complementary strands of DNA provides a basis for identification of biochemical substances such as biomolecules.
With the development of scanning probe microscopes, in particular the atomic force microscope (AFM), surfaces can be probed in physiological environments with molecular resolution and forces down to the piconewton range as is disclosed in an article by G. Binnig, C. F. Quate and Ch. Gerber, Phys. Ref. Lett. 56, 930 (1986).
Using the atomic force microscope, Florin et al. measured the adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin. They found the force required to separate the tip and the bead to be quantized in integer multiples of 160 pN (E.-L. Florin, V. T. Moy and H. E. Gaub, Science 264, 415 (1994)).
It is an object of the present invention to provide a method for identification of unknown substances which requires less effort with respect to necessary equipment and fewer procedure steps. The method should be easy to control and should produce reliable results.
It is a further object of the present invention to provide a device for identification of substances based on it""s binding characteristics to a known probe, in particular comprising a biochemical molecule, without the need of highly sophisticated analyzing devices, which usually are very expensive. Furthermore such a device should be easy to handle and should be used as a biosensor enabling the identification of a plurality of unknown substances.
The present invention relates to a method for identification of a substance, preferably comprising biochemical molecules, which is usually derivatized onto a sample surface. Furthermore, a well-known probe is used which is favorably derivatized onto a contact surface for the purpose of better handling. Both surfaces coated with the aforementioned layers are brought into contact and then separated from each other while measuring physical parameters characterizing the interaction between said substance and said probe. The obtained results are compared with reference values for identification. These reference values can be listed or measured with reference techniques, for example, as is described above in connection with the cited state of the art.
For characterization and identification of an unknown substance, the binding related work of adhesion is a suitable parameter which can be measured with the aforementioned invented method. In a preferred implementation of the invented method a molded elastomeric pillar is used with a well-defined contact surface onto which a known probe is derivatized. This probe is pushed towards the sample surface onto which the unknown substance, preferably biochemical molecules, is derivatized, until both surfaces have a well-defined conformal contact.
The term xe2x80x9cconformal contactxe2x80x9d implies that the surface shapes of the two media put on top of each other are similar to such an extent that fluids can essentially not penetrate into the plane where the surfaces meet each other except for immobilized water strongly associated with the molecules. The term xe2x80x9cfluidxe2x80x9d refers to both liquids and gases.
While both surfaces are brought together until conformal contact is reached the force during this loading phase is determined as a function of the travelling distance from the point of which the two surfaces make the first contact until the state of conformal contact which is indicated by a given force. Subsequently the two surfaces are withdrawn from each other until both surfaces are completely separated. Also the force during the separation phase is determined as a function of the travelling distance from the state of conformal contact until the point of becoming totally separated.
The force applied during the loading phase is summed up from the point of contact until the maximum load. This integral represents the energy stored in the elastic deformation of the pillar. The applied force during the unloading phase is similarly summed up from maximal load to zero load after backlesh compensation.
Surfaces having substances with great work of adhesion have to be pulled away from each other until these snap apart. The difference between the two energies (loading integral, unloading integral) is the work of adhesion and corresponds individually to the substance coated on the sample surface. The obtained value of the work of adhesion can now be compared with a reference value derived from stored data or a reference measurement.
Another alternative method for identification of a substance by obtaining results of high quality and expressiveness about the nature and the type of substance can be reached in the following manner:
As is the case in the aforementioned method the unknown substance is derivatized onto a sample surface which comes into contact with a contact surface onto which a well-known probe is coated. At least one of said surfaces, preferably the contact surface is made of flexible material like elastomeric material so that both surfaces come into conformal contact. Subsequently, both surfaces are withdrawn from each other by applying a defined increasing force to at least one of the surfaces until complete separation between the surfaces is reached. It is important that the involved force which is preferably directed onto the contact surface acts in a predefined manner so that the step of withdrawing can be repeated reversibly and in a standardized manner.
A value which is characteristic for the interaction between the unknown substance and the probe is the time period between commencement of applying the increasing force for withdrawing both surfaces from each other and the point of complete separation. The obtained time period is expressive of the binding forces acting between the substance and the probe and is therefore characteristic of the substance. For identification of the measured substance, the obtained time period has to be compared with a reference value which can be deduced from a stored data set or obtained by typical reference measurements.
Instead of applying a defined increasing force as is described above, in a further alternative method, a defined constant force is applied to at least one of said surfaces for the withdrawing step. Without measuring physical parameters like applied forces or time duration, it is only of interest whether both surfaces having contact or not after applying the constant force onto it.
So it is possible to classify a substance only by its binding behavior. With said constant single force action a threshold value can be determined which helps to make a rough decision whether the to-be-analyzed substance can withstand the applied force or not by checking whether both surfaces stay in contact or not.
Certainly the latter alternative just permits only a rough knowledge about the nature and the type of substance but the method can be seen as a first approach for a rough identification or distinction of substances and can be carried out in a simple way so that this method is suited for field practice.
The invention also relates to a device for identification of a substance, preferably comprising biochemical molecules, with which the inventive method can be carried out. For this, the device comprises basically two carrier means, one for the to-be-analyzed substance and the other one for the known probe. The probe is applied onto a contact surface which is a surface of a support means which preferably is molded of elastomeric material in the shape of a pillar. For a better statistic balancing of determination of the involved binding forces between the substance and the probe, the contact surface of the pillar provides a fraction of xcexcm2 up to several mm2. On the other hand, the unknown substance is derivatized onto a sample surface which can be of rigid material such as a glass substrate.
For bringing into contact as well as for separation of both surfaces, a separation means is provided which preferably is connected with the pillar arrangement so that the pillar can be moved towards and can be withdrawn from the sample surface. In a preferred embodiment the separation means comprises a stepper motor drive which enables a controlled travelling distance between the contact surface of the pillar and the sample surface.
Further measuring means are provided for measuring, for example, applied forces which are directed onto the surfaces for bringing the surfaces into contact or for withdrawing them from each other. Likewise, the measuring means can be designed for determination of the time period between conformal contact of both surfaces and the state of complete separation. A light source might be provided for coupling light into said pillar arrangement for detecting whether the pillar stays in contact to the sample surface or being separated. For this purpose, the pillar has to be made of translucent material so that light can be coupled into the pillar, which works as a waveguide. In the event of having contact between the pillar and the sample surface, the light coupled into the pillar transmits the boundary layer between the contact surface and the sample surface into the body on which the sample surface is provided. Preferably the body of the sample surface is also made of a translucent material so that transmitted light can pass the body without suffering high losses. A detection means is arranged adjacent to the body for measuring the amount of transmitted light.
In the event of having no contact between the contact surface and the sample surface the amount of transmitted light is clearly reduced due to the reflection at the contact surface of the pillar which is surrounded by a medium having a different refraction index compared to that of the pillar material itself. With the detected signal of the light detecting means, for example realized by a CCD camera or more simply by eye, precise information can be obtained about the actual state of both surfaces whether staying in contact or being separated. This signal can be used as a start or stop signal for measuring the aforementioned time it takes to separate both surfaces. Also the light detection means can provide information about reaching the state of conformal contact between the contact surface and the sample surface.
In a further preferred embodiment the inventive device can be used as a biosensor for detection and characterization of a multiplicity of substances in the form of biomolecules. The biosensor comprises at least one pillar made of elastomeric material like polydimethylsiloxane (PDMS) having a diameter from about 0,1 xcexcm to 1 mm and an aspect ratio of about 2-10. The contact surface of the pillar is derivatized with chemical compounds comprises molecules having selective binding properties. These sensing molecules are attached to the contact surface in an oriented way so that the active side of each molecule points away from the pillar surface and can be used for selective sensing. Each molecule is strongly fixed at the contact surface and can be used many times. When the pillar is pressed onto the sample surface and forms a conformal contact, the flexibility of the pillar allows for individual molecules to recognize and bind their ligands in a specific manner typical of biological systems. When the pillar is pulled away from the sample surface, a higher force is necessary than for reference systems without specific recognition. The binding energy can be measured as pointed out above.
In a similar manner, a multiplicity of substances attached on many separate sample surfaces can be tested and classified in an automatic set up when the sample surfaces are fixed onto the bottom of a microtiter plate.
Furthermore, the biosensor can also provide a multiple pillar arrangement, each pillar having a contact surface being coated with one type of biomolecules having different selective binding properties. The pillars of the arrangement are connected by a common connection means so that contact between said contact surfaces of each pillar to a sample surface can occur simultaneously.
The approach described above for measuring biological molecules with that kind of inventive biosensor also works for mixed and dilute systems. In a dilute system the surface of the sensor is only partially covered by the substance to be analyzed. Partial coverage of the sensor surface by the substance to be analyzed can result from a too short exposure of the surface to a solution having this substance, or a small sticking coefficient between the surface and this substance. In a mixed system the substance to be identified is present on the surface of the sensor with other molecules that codeposited from a solution having several types of molecules. To this case the fraction of the sensor surface coated by the substance to identify reflects the relative sticking coefficient and concentration of the various molecules with the surface. In both cases the work of adhesion can be substantially reduced as the density of the substance to be identified on the surface decreases.
A strategy to prevent these effects is the active capture of the substance to identify from the solution to the surface using immobilized capturing molecules. In this strategy the surface of the sensor is first derivatized with a molecule that specifically binds to and immobilizes the substance to identify, concentrating it at the surface of the sensor. The advantage of this strategy commonly used in immun chemistry, for example, is the use of the possible high affinity constant and the high selectivity between the capturing molecule and the substance to identify. In this case the measured work of adhesion remains high even for dilute or mixed solutions.